RECONSTITUTION OF A PROTEIN DISULFIDE CATALYTIC-SYSTEM

Disulfide bonds are important for the structure and stability of many proteins. In prokaryotes their formation is catalyzed by the Dsb prote ins. The DsbA protein acts as a direct donor of disulfides to newly sy nthesized periplasmic proteins. Genetic evidence suggests that a secon d protein called DsbB acts to specifically reoxidize DsbA. Here we dem onstrate the direct reoxidation of DsbA by DsbB. We have developed a f luorescence assay that allows us to directly follow the reoxidation of DsbA. We show that membranes containing catalytic amounts of DsbB can rapidly reoxidize DsbA to completion, The reaction strongly depends o n the presence of oxygen, implying that oxygen serves as the final ele ctron acceptor for this disulfide bond formation reaction. Membranes f rom a dsbB null mutant display no DsbA reoxidation activity. The abili ty of DsbB to reoxidize DsbA fits Michaelis-Menten behavior with DsbA acting as a high affinity substrate for DsbB with a K-m = 10 mu M. The in vitro reconstitution described here is the first biochemical analy sis of DsbB and allows us to study the major pathway of disulfide bond formation in Escherichia coli.